Caldesmon is an actin-binding protein which may modulate actin-based cellular processes. These include activities such as cytokinesis, shape change, cell motility, cell to cell interactions, adhesion to substrata and secretion. In vitro studies have shown that caldesmon can inhibit the actin-activated MgATPase activity of myosin and that this inhibition can be reversed by calmodulin binding to caldesmon. Caldesmon is a substrate for various kinases and has been shown to be phosphorylated during smooth muscle contraction. It is also phosphorylated when human platelets are treated with phorbol esters. We have examined the phosphorylation of caldesmon following treatment of platelets with physiological agonists such as thrombin, ADP and collagen and after treatment with the prostaglandin, PGI2, which raises cAMP levels. There is no increase in the level or sites of phosphorylation of caldesmon following treatment of platelets with thrombin, ADP or collagen, but treatment with PGI2 results in an increase in phosphorylation. Tryptic phosphopeptide maps show that this phosphorylation is primarily occurring at two sites which are also phosphorylated in vitro by cAMP-dependent protein kinase.